This invention relates to a diagnostic method for the direction and determination of antigens and antibodies. More particularly, this invention relates to diagnostic methods for the detection and determination of pathogenic disease antigens and antibodies, such as hepatitis and rubella antigens and their associated antibodies.
A number of immunological methods have been developed for the determination of antigens and antibodies, including methods for the determination of hepatitis B surface antigen (HB.sub.s Ag), a component of hepatitis B virus and its associated antibodies.
In this field it is important that whatever methods are used are as sensitive and reliable as possible since an incorrect diagnosis can have serious consequences. This is especially true of the determination of hepatitis antigens and their associated antibodies since the transmission of the hepatitis virus via blood donors constitutes a significant public health risk.
Up to the present time, the radio-immunoassay (RIA) method in its various forms has been the most sensitive system available. This method has several disadvantages, however, including the requirement of special equipment, trained staff, the need for extra safety measures to protect against harmful radiation, and the short half-life span of the radioactive labelling element. The possibility of replacing the radioactive label with an enzyme label was proposed in 1968 in an article by L. E. M. Miles and C. N. Hales, entitled "Labelled Antibodies and Immunological Assay Systems," Lancet, London 1968 II, page 492; Nature, Vol. 219, pages 186-189 (July 13, 1968), but no procedural details were provided, the article failing to offer more than the general idea, leaving it to future workers to determine the basic steps and to perform the extensive experimentation needed to establish a practical operative enzymic immunoassay method.
The pioneering work on enzyme-immunoassay (EIA) methodology was performed by Schuurs and coworkers, and is disclosed in a series of their U.S. Pat. Nos. 3,654,090, 3,791,932, 3,850,752, 3,839,153, and 3,879,262.
In the course of the further evolution of the radio-immunoassay procedures by numerous workers, it was pointed out out in an article by E. Habermann, entitled "A new Principle for the Quantitative Determination of High Molecular Antigens (Junction Test), etc." published in Z. klin. Chem. u. klin. Biochem., 8th year, January, 1970, pages 51-55, that those methods based on the competition of radio-labelled and unlabelled antigens for a limited amount of antibodies gave better results than other methods.
The Habermann article proposed an RIA method in which in a first step, the antigen is adsorbed on an antibody fixed with a covalent bond by a solid phase, e.g., cellulose; a second step consists in fixing by labelled antibodies those antigen determinants remaining accessible. The non-fixed component of the antibody preparation is removed by washing. The radioactivity of the solid phase is correlated with the antigen content of the original solution. Since the labelled and solid phase antibodies are joined via the antigen, the author called the technique the "junction test." This arrangement is also known in the art as the "sandwich technique or method." The Habermann article, referred to the earlier suggestion of Miles and Hales (loc. cit.), and contained a passing suggestion that the test could be improved in sensitivity by coupling the antibody molecule with an easily detectable enzyme, but also offered no further suggestions, leaving it to others to develop such an enzyme method.
In Schuurs et al. U.S. Pat. No. 3,791,932, there is disclosed, in Example III, a rudimentary sandwich-type procedure for the determination of human chorionic gonadotropin (HCG) and luteinizing hormone (LH) in low concentrations by means of an enzyme-antibody coupling component, in accordance with which a predetermined amount of HCG is first coupled to a solid immuno-adsorbent (m-aminobenzyloxymethyl cellulose). Antibodies from rabbit anti-HCG serum are then coupled to an enzyme (horse radish peroxidase, HRP) and the coupling product is bound to HCG cellulose. HCG and LH dilution series are mixed with anti-HCG cellulose to form an immunoadsorbent, to which a given amount of the antibody-enzyme coupling product is added, and the enzyme activity of the supernatant liquid is determined. This procedure is somewhat involved and requires a number of coordinated operating steps.
It is an object of the present invention to develop and further improve and simplify the early version of the sandwich technique as applied to the determination of antigens and antibodies.